Microbial insecticide

ABSTRACT

An improved microbial insecticide composition, and methods for the production and utilization thereof, are disclosed. The disclosed composition comprises a microbial insect pathogen of viral, bacterial, or fungal origin which is susceptible to sunlight-induced inactivation embedded in a coacervate microbead which is comprised of a nucleic acid, typically RNA, and a proteinaceous material, whereby the microbead structure itself effectively shields the agent from sunlight-induced inactivation. The microbead is typically stabilized by chemical crosslinking.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of application Ser. No. 033,895, filed Apr. 27,1979, now U.S. Pat. No. 4,223,007, which is a continuation-in-part ofU.S. application Ser. No. 835,817, filed Sept. 22, 1977, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to microbial insecticides. More particularly, theinvention relates to a novel microbial insecticide composition and tothe production and utilization thereof.

Microbial insecticides of viral, bacterial, or fungal origin offersignificant advantages over conventional chemical insecticides.Microbial insect pathogens are generally nontoxic and harmless to otherforms of life. In addition, microbial insecticides demonstrate arelatively high degree of specificity, and hence do not endangerbeneficial insects. Moreover, a susceptible insect host is quite slow todevelop resistance to microbial pathogens. Microbial insecticides may beused in relatively low dosages, may be effectively applied as dusts orsprays, and may be used in combination with chemical insecticides.

For example, the Douglas fir tussock moth nuclear polyhedrosis virus(NPV) is a microbial insect pathogen useful for controlling the tussockmoth. Likewise, Bacillus thuringiensis (B.t.), a spore-formingbacterium, is well-known as a microbial insect pathogen useful againstnumerous leaf-chewing insects in their larval stages, including, forexample, alfalfa caterpillars, tomato hornworms, tobacco hornworms,cabbage loopers, cabbage web worms, army worms, gypsy moths, walnutcaterpillars, diamondback moths, cosmopolitan green beetles, Europeancorn borers, and other members of the order Lepidoptera.

Unfortunately, the effectiveness and usefulness in the field of manymicrobial insect pathogens as insecticides are severely limited by theirextreme sensitivity to sunlight. It is known, for example, that one ofthe problems encountered when using B.t. as an insecticide is its shortperiod of effectiveness in the field, which is due, in part, tosunlight-induced inactivation of the microorganism. It is also knownthat nonionizing radiation having a high photon energy (e.g. ultravioletrays) exerts an inactivating effect on B.t.. See, "PhotoprotectionAgainst Inactivation of Bacillus thuringiensis Spores by UltravioletRays," Aloysius Krieg, Journal of Invertebrate Pathology, Vol. 25, pp.267-268 (1975). In particular, it is known that ultraviolet (UV) rayswith a wavelength of 253.7 nm induce a marked, extraordinaryinactivation of B.t. spores, so that they are unable to germinate andgrow out. A dosage of 18 m W sec/cm² of such 253.7 nm wavelengthradiation will inactivate 99.9% of the B.t. spores. However, since UVradiation of wavelengths shorter than about 285 nm do not reach theearth's surface, such inactivation at 253.7 nm is of little practicalconcern in the field.

We have determined that the half life of B.t. subjected to sunlight isapproximately six minutes. Likewise, it has been determined by othersthat the half lives of certain occluded viruses subjected to sunlight isone-half to one hour. Thus, the effectiveness of a typical sprayapplication of such microbial insecticides is rapidly lost in the field.

Since nucleic acids show a maximum of extinction near a wavelength of260 nm, it has been suggested by others that the UV induced death ofB.t. at 253.7 nm, and of certain occluded viruses at comparablewavelengths, may be caused by a photoreaction of the genetic material,especially DNA. Thus, it has been suggested, ibid., at p. 267, that B.t.spores could be protected from inactivation by such UV radiation (253.7nm) by physically mixing the B.t. spores with DNA, or a comparablenucleic acid which would absorb the UV rays. Such a comparable nucleicacid would be RNA, Ribonucleic Acid, which has a maximum of extinctionnear 260 nm. However, this technique proved to be ineffective.Furthermore, as noted above, since wavelengths shorter than about 285 nmdo not reach the earth's surface, the usefulness of DNA or RNA as aprotectant against sunlight-induced (i.e. at wavelengths greater thanabout 285 nm) inactivation is unproven.

SUMMARY OF THE INVENTION

The present invention comprises a microbial insecticide composition andmethods for the production and utilization of such composition.Typically the composition comprises a microbial insect pathogen ofviral, bacterial, or fungal origin which is susceptible tosunlight-induced inactivation embedded in a coacervate microbead whichis comprised of a nucleic acid, typically RNA, and a proteinaceousmaterial, whereby the microbead structure itself effectively shields thepathogen from sunlight-induced inactivation. The microbead is typicallystabilized by chemical crosslinking.

One typical method for preparing the microbial insecticide compositioncomprises: (a) preparing a paste-like mixture comprising (i) nucleicacid particles, (ii) proteinaceous material particles, (iii) microbialinsect pathogens of viral, bacterial, or fungal origin, and (iv) anamount of water sufficient to wet (i.e. hydrate) substantially theentire mixture; and (b) agitating the paste-like mixture in a manneradapted to break up the mixture into discrete microbeads, whereby themicrobial insect pathogens are embedded in the microbeads. Preferablythe discrete microbeads are stabilized by treatment with a chemicalcrosslinking agent such as tannic acid, glutaraldehyde or a similaragent. In one preferred embodiment of the invention the agitation of thepaste-like mixture takes place in a solution containing the chemicalcrosslinking agent.

Another typical method for preparing the composition comprises: (a)preparing an aqueous solution containing a nucleic acid; (b) preparingan aqueous solution containing a proteinaceous material; (c) preparingan aqueous suspension of strongly positively or negativelysurface-charged microbial insect pathogens; and (d) mixing the aqueoussolutions and suspension prepared in steps (a), (b), and (c) together,thereby spontaneously forming microbeads having the insect pathogensembedded therein. In one preferred embodiment the suspension prepared instep (c) is first mixed with the solution prepared in step (a), and thenthis mixture is mixed with the solution prepared in step (b). In anotherpreferred embodiment the suspension prepared in step (c) is first mixedwith the solution prepared in step (b), and then this mixture is mixedwith the solution prepared in step (a).

Typically the surface charge of the pathogens is made strongly negativeor strongly positive by the addition of a protein-modifying agent to abuffered aqueous suspension of the pathogens. The microbeads aretypically crosslinked.

The present invention also comprises a method for controlling insectpests in insect infested areas which typically comprises applying aneffective amount of the insecticide composition described above to theinsect infested areas.

The present invention further comprises the insecticide composition madeby the processes described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the optical density, over the solar UV andvisible range, of two typical types of microbeads suitable for use inthe present invention.

FIGS. 2-12 are graphs showing the comparative experimental data fromExamples 1, 2, and 4-12, below, respectively. In FIGS. 2-4 the number ofviable spores, extrapolated to 1 ml of original sample, is shown as afunction of the length of time of exposure to the UV radiation. In FIGS.5, 6, and 8 the percentage of microbes remaining as survivors is shownas a function of the exposure time. In FIGS. 7 and 10 the number ofviable spores per filter is shown as a function of the exposure time.FIGS. 9, 11, and 12 show LD₅₀ data as a function of the exposure time.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, micriobial insect pathogens of viral,bacterial or fungal origin which are susceptible to sunlight-inducedinactivation are embedded in coacervate microbeads comprised of anucleic acid, typically RNA, and a proteinaceous material. We have foundthat such microbeads provide excellent protection of the pathogensagainst sunlight-induced inactivation. The microbeads act as protectiveshields, serving to intercept and block the harmful radiationwavelengths (i.e. those wavelengths of sunlight which tend to inactivatethe pathogen) before they reach the light-sensitive material of theinsect pathogen.

We have successfully embedded Douglas fir tussock moth nuclearpolyhedrosis virus, Autographa californica nuclear polyhedrosis virus,Bacillus thuringiensis cells, spores, and toxin crystals, and thefollowing vegatative bacterial cells: Pseudomonas fluorescens, Serratiamarcescens, and Escherichia coli in the microbeads of the presentinvention. Based on this, we believe that any microbial insect pathogen,whether of viral, bacterial, or fungal origin, including, but notlimited to, the insect pathogens disclosed in Microbial Control ofInsects and Mites, H. D. Burges and N. W. Hussey, Eds., Academic Press,1971, may be successfully embedded in the microbeads of the presentinvention. Furthermore, based on the experimental data reported in theExamples below, there is little doubt that any insect pathogen embeddedin such microbeads will be protected against sunlight-inducedinactivation.

Various proteinaceous materials may be used in combination with thenucleic acid to form the microbeads, depending on the specific insectpathogen to be protected and the microbead system to be used, including,but not limited to: protamine, cytochrome c, soy protein, hemoglobin,gelatin, synthetic amino acid polymers, etc. In general anyproteinaceous material can be used if the conditions are adjusted so asto facilitate formation of the microbeads. Such conditions may includecharge modification techniques, adjustments in pH, componentconcentrations, etc.

The microbeads in which the microbial insect pathogens are embedded maybe advantageously produced using known techniques for forming what havebeen called coacervate droplets or microbeads. One such technique wasdeveloped in conjunction with the study of the origin of life on earth,and has been used to construct precellular models. See, for example,Evreinova, et al., Journal of Colloid and Interface Science, Vol. 36,No. 1 (1971). According to this technique, an aqueous solutioncontaining a nucleic acid (preferably ribonucleic acid, RNA) and, ifnecessary, a buffering agent (e.g. sodium phosphate or sodium acetate)designed to maintain the pH at a position which optimizes the charge onthe nucleic acid, the protein and the microbe, e.g. with respect to thedesired pI (where the microbe is sensitive to pH, this should be takeninto account as well), is mixed with an aqueous solution containing anappropriate proteinaceous material. Protein-nucleic acid microbeadswhich are essentially solid and roughly spherical form spontaneouslyupon the mixing of these two solutions. In the discussion which follows,the above-described technique will be referred to as the "solutionformulation" technique, and microbeads formed according to thistechnique will be referred to as "solution formulation" microbeads.

In an equally preferred embodiment of the invention the microbeads inwhich the microbial insect pathogens are embedded may be produced usinga new technique which we have developed. In the discussion whichfollows, this new technique will be referred to as the "pasteformulation" technique, and microbeads formed according to thistechnique will be referred to as "paste formulation" microbeads.According to this new paste formulation technique, a paste-like mixtureis prepared comprising nucleic acid (preferably RNA) particles,proteinaceous material particles, and an amount of water sufficient towet substantially the entire mixture, and then this paste-like mixtureis agitated in a manner designed to break up the mixture into discretemicrobeads. Such agitation may be accomplished by conventionaltechniques such as, for example, rapid stirring or blending (in aconventional blender), sonification, shaking, pressure extrusion, etc.In order to facilitate the breaking up of the mixture into discretemicrobeads, it may be desirable, in certain embodiments, to extrude thepaste-like mixture into filaments, pellets, etc. prior to agitation. Itis generally preferred to stabilize the discrete microbeads by treatmentwith a chemical crosslinking agent (e.g. tannic acid, etc.) eitherduring or after agitation. However, crosslinking may be unnecessarywhere stabilization may be effected by other means, such as, forexample, freeze drying.

With respect to the solution formulation technique, and to a certainextent the paste formulation technique, it should be noted that whileall of the above-named proteinaceous materials, and others, can be usedsatisfactorily in forming the microbeads, care must be taken to maintainthe pH of the mixture of solutions on the acid side of the isoelectricpoint of the particular protein being used, since this is required forformation of the microbeads. For example, when using protamine the pHshould be maintained below about 11, and when using hemoglobin the pHshould be maintained below about 7. Furthermore, if a protein is usedthat is insoluble at a given pH, the pH may have to be put in a range inwhich the protein is soluble, or other steps may have to be taken tomake the protein soluble. These steps could include partial degradation,charge modification or adding other components in the buffers (e.g.detergents, alcohols, surfactants, etc.). If foaming of one or morecomponents is a problem, simethicone type agents (U.S. Pat. No.2,441,098) can be included. When RNA is being used as the nucleic acidin the solution formulation technique, the pH of the mixture ofsolutions must be maintained at or above about 4.3 to prevent the RNAfrom precipitating out of the microbead, with the protein necessarilyleaving the microbead and going back into solution.

As can be seen, in the above-described solution and paste formulationtechniques for forming microbeads (i.e., coacervate droplets), thematerials to be utilized to intercept and absorb the harmful radiationform the microbead structure, thereby producing a higly protectivecoating.

The bimolecular structure of the microbeads creates a thermodynamicallystable cooperation between the components, so that even withoutsubsequent chemical crosslinking, as described below, the componentswill not individually diffuse out of the microbeads. The bimolecularstructure also causes the microbeads to be highly charged. These chargesshould aid the microbeads in sticking to plant surfaces. These chargescan be controlled by selecting the appropriate protein to be used informing the microbead.

It has been reported that, the size of the microbeads (solutionformulation) can be controlled by controlling the concentration of thenucleic acid and the protein in the formation vessel. We havedetermined, for example, that 100μ diameter solution formulationmicrobeads can be made by mixing an equal volume of 5% RNA and 10%protamine sulfate. Microbeads will form and settle to the bottom of thevessel. Most of these will be in the 100μ range. We have also found thatthe size of the microbeads can be controlled, to some extent, by thedegree of agitation during formation, with greater agitation producingsmaller microbeads. While microbeads having an effective diameter withinthe range of from about 10 to about 200 microns should be suitable foruse in the present invention, it will generally be preferred to utilizemicrobeads having an effective diameter within the range of from about40 to about 100 microns. In general, the type of vegetation (i.e. crops,trees, etc.) to be treated and the method of application will determinethe desired microbead size.

While a relatively wide range of nucleic acid and proteinaceous materialconcentrations can be used to make these microbeads generally, inpreparing microbeads for use in the present invention (i.e. forentrapping microbial insect pathogens) it is preferred to use a nucleicacid: protein ratio in the range from about 1:5 to 5:1.

In those embodiments of the invention wherein it is desired to utilizethe solution formulation microbeads described above, the microbialinsect pathogens may be embedded (i.e., entrapped) in the microbeads bysimply placing them in suspension in water (a buffering agent, e.g.phosphate, acetate, etc. may optionally be added if necessary to controlpH) and then mixing this suspension with an aqueous solution containingthe desired nucleic acid. The resulting suspension is then mixed withthe aqueous protein solution as described above and the pathogen isspontaneously embedded in the proteinaceous material-nucleic acidmicrobeads which form. As a less preferred alternative procedure, thebuffered suspension of microbes may be first mixed with theproteinaceous material solution and then the resulting suspension mixedwith an aqueous solution containing the nucleic acid. It has been foundthat subsequent shaking of the vessel in which the solutions have beenmixed will cause the microbeads to coalesce and spontaneously reform,usually resulting in additional pathogens being embedded in themicrobeads.

In those embodiments of the invention wherein it is desired to utilizethe paste formulation microbeads described above, the microbial insectpathogens may be embedded in the microbeads by simply placing them insuspension in water (as above, a buffering agent may be added asnecessary) and then mixing this suspension with the mixture of nucleicacid particles and proteinaceous material particles (care should betaken to use only an amount of water sufficient to wet the mixture andgive it a paste-like consistency). The resulting paste-like mixture isthen agitated as described above so as to break it up into discretemicrobeads. As an alternative procedure, nucleic acid particles andproteinaceous material particles may be mixed with an amount of watersufficient to wet the mixture and give it a paste-like consistency, andthen the microbial insect pathogens may be mixed with this paste-likemixture and embedded in discrete microbeads by agitation of the mixtureas described above.

While the microbeads produced according to the abovedescribed solutionformulation and paste formulation techniques possess a certain degree ofstability, it will generally be advantageous to increase their stabilityto faciliate separation of embedded pathogens from non-embeddedpathogens and to further facilitate handling. This is particularly sowith respect to paste formulation microbeads. In a preferred embodimentof the invention, such stabilization is accomplished by chemicallycrosslinking the microbead molecules by treating them with crosslinkingagents such as, for example, tannic acid, glutaraldehyde, imidoesteragents, dithiobissuccimidyl propionate, etc. using conventionalcrosslinking techniques. If it is desired to use glutaraldehyde, anaqueous solution of 0.25%, or less, (by weight) should be used, since wehave found that as the glutaraldehyde concentration is increased,certain pathogens, in particular, Bacillus thuringiensis, will tend tobecome inactivated. We have found that buffered tannic acid is non-toxicto bacterial spores at a concentration of 10% (w/v), and we believe thatit will be non-toxic to most microbial insect pathogens atconcentrations of 1% or less (w/v). While buffered tannic acid having aconcentration within the range of from about 0.001% to 10% should besuitable for use, concentrations within the range of from about 0.5% to1.5% will generally be preferred.

It should be noted that the depth of crosslinking can be controlledrather easily by controlling the time, concentration, temperature, andother conditions of crosslinking. For example, the depth of crosslinkingmay be controlled by stopping the crosslinking reaction by adding asmall molecule which reacts with the crosslinking reagent (e.g. lysineadded to glutaraldehyde) or by using low crosslinking reagentconcentrations.

Such chemical crosslinking of the microbeads yields several advantages,including: (1) stabilization against the shear forces created by sprayapplication of the insecticide; (2) maintenance, if desired, of fluidcenters within the microbeads; (3) maintenance, if desired, of a pHlevel inside the microbead which is lower than that of the environmentsurrounding the microbead (i.e., alkaline digestive juices of the insectgut) so that the interior of the microbead may be kept at a pH valuenear the optimum pH value for viability, storage, etc. of the microbialpathogen; (4) control of the position in the insect gut where thepathogen is released (i.e. the greater the crosslinking, the furtheralong in the gut release will occur and vice-versa), thereby increasingthe infectivity of the pathogen. In addition, the use of tannic acid asthe crosslinking agent increases the optical density of the resultingcrosslinked microbeads (see FIG. 1), thereby providing improvedshielding of the embedded microbes against sunlight-inducedinactivation.

We have also found that the microbial insect pathogen may be embedded(i.e. entrapped) in the above-described solution formulation microbeadsmuch more readily and in much greater numbers if its net surface chargeis first modified so as to be made nearly totally (i.e. strongly)negative or nearly totally positive. We believe this will also be thecase with regard to paste formulation microbeads. This surface chargemodification may be accomplished, for example, by the controlledaddition of a protein modifying agent such as, for example, succinicanhydride (to make strongly negative) and similar compounds (see e.g.,Gary E. Means and Robert E. Feeney, Chemical Modifications of Proteins,Holden Day, Inc., 1971). However, care must be taken to select aprotein-modifying agent which will not inactivate or harm the pathogento be embedded. For example, we have found that succinic anhydride isnot suitable for use with vegetative bacterial cells (e.g. Serratiamarsescens, etc.), since it tends to inactivate these cells.Modification to a strongly positive surface charge may be accomplished,for example, by using tannic acid to link positively charged proteins(e.g. protamine) to the surface of the pathogen. We have found, incomparative studies, that in the absence of any charge modification,only about 1% of the available B.t. cores are embedded in the microbeads(solution formulation), and that using strongly negatively surfacecharged B.t. spores (succinic anhydride treatment) results in about10-20% of the B.t. spores being embedded in the microbeads, and thatusing strongly positively surface charged B.t. spores (treatment withtannic acid then protamine sulfate) results in about 20-40% of the B.t.spores being embedded in the microbeads. The effectiveness of thesecharge modification techniques may be increased by first washing themicrobial insect pathogen, and it may be desirable, in certainembodiments, to wash in separate organic (e.g. 60% ethanol solution, byweight) and inorganic (e.g. 1 M sodium chloride solution) washes,provided the pathogen being used is not sensitive to such materials. Wehave found that, prior to attempting any of the above-described chargemodification techniques on insect viruses, it will generally bepreferred to purify the viruses (e.g. by centrifugation, filtration,etc.) in order to remove the insect debris.

Since the charge-modified pathogen apparently competes with thelike-charged component of the microbead for positions in the bead, itmay be necessary to reduce the concentration of such like-chargedcomponent to a level which will faciliate incorporation of the pathogeninto the microbead. For example, if it is desired to entrap microbialinsect pathogens which have been modified to a strongly negative surfacecharge in an RNA-protein microbead as described above, it may benecessary to reduce slightly the concentration of the RNA solution (RNAis also negatively charged) prior to mixing with the protein solution.

Likewise, in such a microbead system, if the surface charge of thepathogen has been modified to a strongly positive surface charge, thenit may be necessary to reduce slightly the concentration of the proteinsolution (protein is positively charged) prior to mixing with the RNAsolution. This latter system may be more attractive for purposes of thepresent invention since it does not require reduction of the amount ofradiation-absorbing material (i.e. RNA).

Since a primary object of the present invention is the protection oflight-sensitive microbial insect pathogens against inactivation byharmful radiation from the sun, it is obviously important, in selectingthe materials to be used in formulating the microbeads, to selectmaterials which strongly absorb and/or reflect such harmful radiation.Preferred materials for use in constructing the microbeads are RNA(ribonucleic acid) and a proteinaceous material such as, for example,hemoglobin, protamine, or a synthetic amino acid polymer. The opticaldensity (i.e. absorption+reflection) of the microbeads comprised of RNAand protamine, produced according to the above-described solutionformulation technique, is shown in FIG. 1. The solid line in FIG. 1shows the optical density of a solution of microbeads made by combiningequal volumes of 0.33% RNA and 0.5% Protamine (crosslinked withglutaraldehyde) over the solar UV range. The dashed line in FIG. 1 showsthe optical density of a solution of microbeads made by combining equalvolumes of 0.67% RNA and 1% Protamine (crosslinked with tannic acid)over the solar UV and visible (to 600 nm) range.

In many cases the presence of a nucleic acid in the microbead will offera second advantage. It has been suggested that the damage caused bywavelengths of sunlight greater than 313 nm is, in the case of manymicrobes, primarily the result of the reaction of the microbe's nucleicacids with free radicals (it is believed that radiation damage totyrosine produces H₂ O₂ which, in turn, produces free radicals). Thenucleic acid present in the microbead structure will tend to reactspecifically with the free radicals which would otherwise react with themicrobe's nucleic acids, thus preventing any damage.

Since the pathogenic effect of the microbial agent cannot be realized solong as the agent remains embedded within the microbead, care must betaken in selecting the materials to be used in formulating themicrobeads to select those which will permit release of the agent afteringestion of the microbeads by the insect. While other materials mightbe selected, we have found that microbeads comprised of a protein and anucleic acid (e.g. RNA) provide quite satisfactory releasecharacteristics.

After ingestion of the microbeads by the insect, the microbeads will beattacked by proteases and nucleases in the insect digestive tract (i.e.,gut), which will lead to release of the microbe. Thus, it is importantto select microbead materials which are not resistant to such type ofattack. We have found that if Bacillus thuringiensis (cells, spores andtoxin crystals) embedded in microbeads comprised of RNA and protamine(produced according to the above-described solution formulationtechnique) are incubated at room temperature in the presence of insectdigestive juices, release of the B.t. begins within minutes, withprogressive and complete release following within one half hour.

We have also found that if Bacillus thuringiensis embedded in microbeadscomprised of RNA and protamine (produced according to theabove-described technique) are incubated at room temperature in thepresence of amino acids and/or sugars such as would be found in aninsect digestive tract, the germinating spores themselves dissolve themicrobeads in approximately two hours. This does not occur in water orbuffer alone, so that the microbeads will remain intact on leafsurfaces.

Furthermore, the experimental data shown in FIGS. 9, 11 and 12 indicatethat Douglas fir tussock moth NPV viruses and Autographa californica NPVviruses embedded in the microbeads of the present invention are, infact, released in the insect gut, and that upon being so released theyexert a killing effect as desired.

It should be noted that the present invention is suitable for use withany light sensitive microbial insect pathogen, including those of viral,bacterial, or fungal origin. Examples 1-5, 7 and 10 below, illustratethe applicability of the present invention to a typical spore-formingbacteria (i.e. B.t. cells, spores, and toxin crystals), and Example 8,below, illustrates the applicability of the invention to three speciesof vegetative bacterial cells. Example 6, shows the applicability of thepresent invention to a bacterial virus. The positive results shown inExample 6 indicate that the present invention should be suitable for usein protecting non-occluded insect viruses against sunlight inducedinactivation. Examples 9, 11 and 12 illustrate the applicability of thepresent invention to two species of occluded insect viruses.

In the Examples (and Figs.) which follow, the microbial insect pathogenswhich are labelled and/or referred to as "unprotected" comprisepathogens which were not embedded in microbeads. In each example the"unprotected" pathogens were treated, exposed, and tested for viabilityin a manner as nearly identical as possible to the pathogens which wereembedded in microbeads (i.e. the "unprotected" pathogens constitutedcontrol experiments).

The following Examples illustrate several different embodiments of thepresent invention. It is intended that all matter in these Examples andin the foregoing description of the preferred embodiments andaccompanying drawings be interpreted as merely illustrative and not in alimiting sense.

EXAMPLE 1

1×10⁹ spores of Bacillus thuringiensis, including bacterial cells,spores and asporal (crystalline) bodies, obtained from a sporulationmedium culture, were mixed in 10 ml of a 0.15 N phosphate buffer at pH7.5. 1.5 ml of this solution was mixed with 1.5 ml of a buffered 1.34%aqueous solution (by weight) of yeast RNA (obtained from Sigma as gradeB). Then 0.4 ml of this suspension was mixed with constant stirring in1.9 ml of a buffered 0.36% aqueous solution (by weight) of protaminesulfate (obtained from Sigma as grade B).

RNA-protamine microbeads formed spontaneously, each entrapping some ofthe bacterial cells and/or spores and/or asporal bodies. Shaking themixture resulted in breakage and subsequent spontaneous reformation ofadditional microbeads. The microbeads were placed in a glass petri dishand exposed to a General Electric G30T8 30 watt germicidal lamp. Thepetri dishes were placed on a rotary shaker 78 cm below the lamp andshaken at 40 rpm. Viability was determined by plating on brain heartinfusion agar obtained from Difco.

When exposed to germicidal ultraviolet radiation (peak radiation at 254nm) sufficient to kill 99.99% of any unprotected B.t., the B.t. whichwas embedded in the microbeads (i.e., the protected bacterial cellsand/or spores and/or asporal bodies) nearly all survived. This is shownin FIG. 2. The early die-off shown by the line marked "protected B.t."is thought to be due to the low percentage of B.t. actually embedded inthe microbeads. Under microscopic observation the percentage of B.t.actually embedded was observed to range from 0.5% to 1.5%.

EXAMPLE 2

Microbeads with B.t. embedded therein were prepared as in Example 1, andthen 1 mg/ml dithiobissuccimidyl propionate in DMSO was added tocrosslink and stabilize the microbeads. 0.2 ml of this solution wereplaced on a 0.22μ Millipore filter and allowed to dry under vacuum. Thefilters were exposed as in Example 1 without shaking. After shaking, thefilters were washed off in dilution buffer and plated as in Example 1.Results of this procedure are shown in FIG. 3.

EXAMPLE 3

1×10⁹ spores of B.t. obtained from culture in sporulation medium werefirst washed in a 60% ethanol solution and then washed in a 1 M NaClsolution, with the B.t. being separated from these washes bycentrifugation. The washed B.t. was then suspended in 20 ml of a 1 Msodium carbonate buffer solution at a pH of 8.0.

Next, dry succinic anhydride, a protein modifying agent, was added tothe suspension as six separate additions of 2.5 mg/ml each. Theadditions were made under constant stirring and the mixture was stirredfor 10 minutes between each addition. The pH was held at 8.0±0.1 byaddition of NaOH. When the reaction was completed, as indicated by thepH ceasing to change, the B.t. was separated out (centrifuged) andwashed.

This modified B.t. (negatively charged) was then incorporated intoRNA-protamine microbeads according to the procedures set forth inExample 1 and the microbeads were crosslinked as in Example 2.

It was found that this modified B.t. entered the microbeads much morereadily and in much higher numbers than the unmodified B.t. used inExamples 1 and 2. Presumably this was due to the modification of thesurface charge on the B.t. from neutral to negative.

EXAMPLE 4

A solution of unprotected B.t. (cells, spores, and toxin crystals) andprotected B.t. (i.e., embedded in microbeads as described in Example 3,but without crosslinking), 60% unprotected and 40% protected (determinedmicroscopically), was subjected to 254 nm radiation as described inExample 1.

Essentially no unprotected spores remained viable after 15 minutes ofsuch irradiation, while 1×10⁶ protected spores (i.e., 40% of the totaloriginal mixture) remained viable after one hour of such irradiation.Viability was determined as in Example 1. The results of this experimentare shown in FIG. 4.

EXAMPLE 5

A concentration of 1×10⁹ spores of Bacillus thuringiensis (includingcells, spores and asporal crystals) of B.t. was suspended in 10 ml of0.15 N phosphate buffer, pH 7.5 (B.t. preparation was obtained andmodified as in Example 3). 0.5 grams of RNA (Calbiochem, grade B) wasdissolved into this suspension and mixed by vigorous mixing in a Vortexmixing device. The suspension was then added to a buffered 10% solutionof protamine sulfate (Calbiochem, grade B, by weight) and vigorouslyshaken for 5 seconds. Glutaraldehyde (25%, from Sigma) was added to thesolution to a final concentration of 0.15% (by volume). After 30 minutesa pellet formed at the bottom of the tube which consisted of largemicrobeads (100-150μ). The supernate was drawn off and the pellet wasresuspended to a final volume of 20 ml by shaking. This solution wasplaced on a 0.22μ Millipore filter and dried overnight under vacuum. Thefilters were exposed to sunlight (1:00 pm, RH 23%, temperature 89° F.).The filters were then washed in acetate buffer (0.15 N, pH 4.0) to breakup the microbeads and release the B.t., which was plated as in Example1.

The results of this experiment are shown in FIG. 5. Unprotected sporeswere nearly all killed after 30 minutes.

EXAMPLE 6

The purpose of this example was to demonstrate protection of a virusaccording to the present invention. The reactions and responses of aninsect virus and a bacterial virus (called bacterial phage) should besimilar since both are composed basically of a nucleic acid in a proteincoat. Accordingly, we chose to model our system with the bacterial virusof E. coli, phage T-4.

T-4 bacterial phages were grown in nutrient broth with 0.5% NaCl(P-broth). E. coli BB was inoculated into 100 ml P-broth and allowed togrow overnight. In the morning a 1:100 dilution was made to fresh brothand growth was allowed to proceed for one hour. 1×10⁷ phages were addedto this rapidly growing E. coli BB culture and allowed to grow for sixhours (37° C., rapid shaking). At the end of the period, 5 drops ofchloroform were added to kill all bacteria in the culture. This is thephage stock.

Microbeads were prepared by mixing 0.100 grams of protamine sulfate(Calbiochem, grade B) in 10 ml phage stock. This suspension was added to1% RNA (by weight) in P-broth. The microbeads formed spontaneously. TheUV exposure was carried out as in Example 1. Timed samples were takenand dilutions were made in P-broth. The viable phages were determined bythe method described in the following text: Grace C. Rovozzo and CarrollN. Burk, A Manual of Basic Virological Techniques, Prentice-HallBiological Techniques Series, 1973, page 168, using P-broth agar and E.coli BB as the indicator bacteria.

The results of this experiment are shown in FIG. 6. These data show thatunprotected virus were all killed in approximately 5 minutes. On theother hand, after an initial drop similar to that seen in the bacterialtests, the virus which was embedded in the microbeads show strong UVlight resistance (approximately 40% are protected from inactivation).

EXAMPLE 7

Microbeads having B.t. (cells, spores, and toxin crystals) embeddedtherein were prepared as in Example 3, except that dithiobissuccimidylpropionate in DMSO was not used to crosslink the microbeads. In thisexample the microbeads were crosslinked and stabilized by addingphosphate-buffered tannic acid (1% w/v) to the microbead suspension, andallowing it to stand at room temperature for about 30 minutes. Thesuspension of crosslinked microbeads was diluted with dilution buffer(phosphate) in a manner selected to produce a diluted suspensioncontaining approximately 100 spores per ml, and 1 ml samples of thisdiluted suspension were pulled onto separate 0.22μ Millipore® filtersand permitted to dry overnight. The filters were exposed under a GeneralElectric sunlamp measured at 1572 watts/m² at 15 inches and providingradiation in the wavelength range of about 290 nm to 400 nm. One hourexposure under this sunlamp was equivalent to approximately 151 hoursunder direct sunlight on a July day at a location of approximately 45°latitude (i.e. about 15 days). After exposure, the spores were placeddirectly on plate count agar, and after 24 hours incubation the coloniesgrowing on the filters were counted. The experimental data are shown inFIG. 7.

EXAMPLE 8

Vegetative bacterial cells of the species Pseudomonas fluorescens,Serratia marcescens, and Escherichia coli were treated with succinicanhydride, in three separate series of experiments, by placingapproximately 1×10⁹ cells in 20 ml of 1 M carbonate buffer solution (atpH 8.0), then adding 50 mg of succinic anhydride. The pH was notmaintained because the resultant pH, about 7.5, was believed to be moreadvantageous for bacterial survival during harvesting and washing. Thesuccinic anhydride treated cells of the three species were suspended,again in separate tests, in 4 ml of a 0.15 N phosphate buffer solution(at pH 7.5), and 1 ml of this suspension was then mixed with 1 ml of abuffered 1.34% (w/v) RNA (obtained from Sigma) solution. The resultingsuspension was mixed with 2 ml of a buffered 1% (w/v) Protamine sulfate(obtained from Cal Biochem.) solution, spontaneously forming themicrobeads, and then 0.4 ml of a 10% (w/v) tannic acid solution wasadded to the suspension of microbeads. (Note: the buffer referred toabove was 0.15 N phosphate buffer solution at pH 7.5.) The suspension ofmicrobeads in tannic acid was allowed to stand for 30 minutes at roomtemperature, and then was washed three times in the phosphate buffersolution described above by centrifuging.

Microscopic observation revealed that approximately 30% of each of thethree species tested were embedded in the microbeads.

The suspensions of microbeads produced in the three series ofexperiments were exposed, separately, under the General Electric sunlampdescribed in Example 7 using the experimental procedure described inExample 1 (i.e. exposure while on a rotary shaker), except that agreater number of microbeads were present in the suspension. Data from arepresentative test of the P. fluorescens bacterial cells are shown inFIG. 8. The data from the tests of the Serratia marcescens andEscherichia coli bacterial cells (not shown) were similar to that shownin FIG. 8.

It should be noted however, that in performing the experiments for whichdata are shown in FIG. 8, it was assumed that any bacterial cells in themicrobead suspension which had not been embedded in the microbeads wouldbe killed during exposure of the suspension while on the rotary shaker.To test the correctness of this assumption a second control experiment(in addition to the control shown in FIG. 8) was run. In this secondcontrol, B.t. spores were treated as described above, except that theywere not embedded in microbeads, but were merely mixed in a suspensioncontaining already-formed microbeads (using approximately the samenumber of microbeads as used in the experiments described above), andthis mixture was exposed under the sunlamp as described above. If theassumption was correct, then most of the cells should be killed. Wefound, however, that the cells were killed at roughly the same rate asthe cells which had been embedded in the microbeads. We attribute thisto the relatively large number of microbeads present in the suspensionswhich were exposed (i.e. the microbeads suspensions were too heavy). Webelieve that the large number of microbeads present restricted themovement of the cells which were outside the microbeads, so that anycells which were underneath a microbead essentially remained therethroughout the exposure and were protected. Thus, we feel that thisexample, including the second control, does show that the microbeadsprotect the cells against sunlight-induced inactivation (whether thecells are in or outside, but under, the microbeads). Also, as notedabove, microscopic observation revealed that cells of each of the threebacterial species tested were embedded in the microbeads.

EXAMPLE 9

1×10⁸ Douglas fir tussock moth nuclear polyhedrosis virus was suspendedin a 0.5 N phosphate buffer (20 ml) at pH 7.5 and treated with six 50 mgadditions of succinic anhydride. 1.0 NaOH was used to hold the pH at7.5. After treatment, the virus suspension was harvested bycentrifugation and washed twice in 0.15 N phosphate buffer andresuspended in 1 ml of the buffer. This suspension was then treated asdescribed in Example 8 to form crosslinked microbeads having the virusembedded therein. The suspension was exposed under the General Electricsunlamp as described in Example 8, then diluted with buffer and testedfor infectivity as described in Example 12. The infectivity data isshown in FIG. 9. This data suffers the same error as described inExample 8 (i.e. microbead suspension which was exposed was too heavy topermit kill off of virus which were not embedded in the microbeads).However, as in Example 8, microscopic observation did reveal that about30% of the virus was embedded in the microbeads, and that the microbeadsoffered protection against sunlight-induced inactivation (whether thevirus are inside or outside, but under, the microbeads).

EXAMPLE 10

1 gram of hemoglobin (crude powder) was mixed with 0.34 gram of RNA indry powder form. Approximately 1×10⁹ spores of B.t. were suspended in abuffer solution (0.15 N acetate, pH 5.0), and then added to theRNA-hemoglobin mixture. Additional buffer solution was added in anamount sufficient to form a hydrated (i.e. wet) mass having a paste-likeconsistency. The total amount of buffer solution mixed with theRNA-hemoglobin mixture was about 1 ml. This paste was forced (i.e.extruded) through an 18 gauge needle (using a 5 cc syringe) into abuffered (0.15 N acetate) 1% (w/v) tannic acid solution and subjected tomagnetic stirring in a beaker. After about 30 minutes of stirring, theextruded paste had been broken into discrete, crosslinked microbeadsapproximately 100μ or smaller in size having the B.t. incorporatedtherein. The microbead suspension was diluted and exposed under aGeneral Electric sunlamp as described in Example 7 (i.e. on filters).The experimental data are shown in FIG. 10.

EXAMPLE 11

Autographa californica nuclear polyhedrosis virus (approximately 1×10⁷polyhedral inclusion bodies) was embedded in RNA-Protamine microbeadsusing the technique described in Example 10. In this example, 1 gram ofprotamine (dry powder form) and 0.67 gram of RNA (dry powder form) wereused, the buffer was 0.15 N phosphate solution at pH 7.5, and tannicacid was used to crosslink and stabilize the microbeads. The microbeadsuspensions were washed twice in distilled water and then diluted 1:100in distilled water. The diluted suspensions were exposed under a GeneralElectric sunlamp using the technique described in Example 1, and theexposed suspensions were diluted in phosphate buffer as needed for theinfectivity tests, described below. Trichoplusia ni larvae were used tomeasure infectivity of the protected virus (i.e. embedded in themicrobeads) and the unprotected virus (i.e. the control). Infectivitywas determined by placing 10 μl of separate dilutions (1:10² to 1:10⁷)on a 0.2 gram piece of diet. The larvae were allowed to eat the entirepiece of diet, and then a new piece of diet (2 grams) was placed in thevial. When all of the control larvae (not fed virus microbeads) hadpupated, the dead larvae were autopsied to verify that death was causedby infection caused by the Autographa californica virus (this was foundto be the case). LD₅₀ 's for the T. ni. larvae were determined for viruswhich had been protected by the microbeads and for the control virus,which had not been protected by any microbeads. The general proceduresfor making LD₅₀ determinations are described in Microbiology, at p. 639,B. D. Davis, et al., Harper and Row, 1973. The LD₅₀ data are shown inFIG. 11.

EXAMPLE 12

Example 11 was repeated using Douglas fir tussock moth nuclearpolyhedrosis virus in place of the Autographa californica virus andDouglas fir tussock moth larvae in place of the T. ni. larvae, exceptthat the larvae were autopsied at the end of a 10 day period. The LD₅₀data are shown in FIG. 12.

It should be understood that the term "nucleic acid" as used throughoutthis specification and in the claims is intended to include allpolynucleotides. Likewise, the term "protein" is intended to include allpolypeptides.

It is also to be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and such modifications andvariations are considered to be within the scope of this invention asdefined by the appended claims.

We claim:
 1. An insecticidal composition comprising a microbial agent incombination with at least one of its biologically active chemicalproducts embedded in a coacervate microbead having an effective diameterwithin the range of from about 10 to about 200 microns which iscomprised of a nucleic acid and a proteinaceous material admixed in anucleic acid to proteinaceous material ratio in the range of from about1:5 to about 5:1.
 2. The insecticidal composition of claim 1, whereinthe microbead is further stabilized by chemical crosslinking.
 3. Theinsecticidal composition of claim 1, wherein the nucleic acid comprisesribonucleic acid.
 4. The insecticidal composition of claim 1, whereinthe proteinaceous material comprises hemoglobin, protamine, or asynthetic amino acid polymer.
 5. The insecticidal composition of claim1, wherein the effective diameter of the microbead is within the rangefrom about 40 to about 100 microns.
 6. An insecticidal compositioncomprising a microbial agent in combination with at least one of itsbiologically active chemical products, said microbial agent and chemicalproducts being susceptible to environmentally-induced inactivation andbeing embedded in a coacervate microbead having an effective diameterwithin the range of from about 10 to about 200 microns which iscomprised of a nucleic acid and a proteinaceous material admixed in anucleic acid to proteinaceous material ratio in the range of from about1:5 to about 5:1, whereby the microbead structure effectively shieldssaid agent and chemical products from environmentally-inducedinactivation.
 7. The insecticidal composition of claims 1 or 6 whereinthe microorganisms and chemical products possess an essentially uniformsurface charge when they are embedded in the microbead.
 8. A method forcontrolling insect pests in insect-infested areas, which comprisesapplying an effective amount of the insecticide composition of claims 1or 6 to said insect-infested areas.